Project Summary Human aging results in cellular damage and a decline in function, creating a significant risk factor for many diseases, including cancer. One long-standing theory of aging, the Mitochondrial Free Radical Theory of Aging (MFRTA), attributes much of age-associated cellular damage to the main reactive oxygen species (ROS) producers in cells: the mitochondria. To prevent direct exposure to ROS, mitochondrial DNA (mtDNA) is packaged by proteins, forming nucleoprotein complexes called nucleoids. However, little is known about the material properties of mitochondrial nucleoids and how those properties contribute to function during aging. The long-term objective of this work is to determine how the physical properties of mitochondrial nucleoids influence nucleoid self- organization as well as mitochondrial function in the context of aging. Here, equilibrium thermodynamic principles governing liquid-liquid phase separation will be applied to describe the behavior of aging mitochondrial nucleoids. The principal hypothesis is that mitochondrial nucleoids represent a separate phase of nucleoprotein complexes that are physically distinct from the surrounding mitochondrial matrix, analogous to how oil and water de-mix. By extension, age- associated damage to nucleoids is expected to alter the material properties of mitochondrial nucleoids, ultimately impairing mitochondrial function and contributing to the disease phenotype. The specific aims are: 1) to analyze how aging drives condensation (phase separation) of mtDNA; 2) to determine the molecular interactions controlling nucleoid phase behavior; and 3) to investigate the role of phase separation on mtDNA protection and function. Fibroblasts derived from young, old, and prematurely aging patients will be used as a model system. Methods include several light microscopy techniques to visualize mitochondrial nucleoids, accompanied by quantitative image analysis and physical modeling of the phase behavior. Additionally, a focused RNAi screen of known nucleoid proteins will identify molecular interactions that are responsible for controlling phase separation, while a battery of functional assays will be used to compare nucleoid condensation/dissolution state with mitochondrial function during aging. The significant contribution of this work is to elucidate the phase behavior of mitochondrial nucleoids and functional consequences during aging, thereby directly testing the MFRTA. This interdisciplinary approach will bridge the physical self-assembly of mitochondrial nucleoids with function and disease, revealing the biophysical mechanisms that describe the normal aging process.